The goal of this renewal proposal continues to be the development of novel, sensitive electrochemical sensors for DNA diagnosis. We propose to optimize our DNA electrochemistry assay for mutational analysis, to design novel electrochemical sensors for nucleic acid sequence detection, to develop and apply DNA modified electrodes to analyze nucleic acid and DNA-protein structures and their associated redox chemistry. Fundamental to the design of these new DNA-based sensors are long-range DNA-mediated charge transport (CT) chemistry and its sensitivity to perturbations in DNA structure and base pair stacking. Specifically we plan to optimize our electrochemical sensor for single base mutations to obtain robust, reproducible and sensitive detection starting from genomic DNA samples. After annealing and assembling test samples in solution, processed samples will be addressed to the electrochemical chip utilizing sticky-end overhangs for addressing. The length of DNA targets will be varied, multiplexed addressing will be explored, and different biochemical methods for the preparation of test samples will be optimized. Graphite will also be probed as an alternate electrode surface. With the aim of constructing a sensitive electrochemical sensor for pathogen detection, our basic DNA CT strategy for mutation detection will be modified and expanded to allow the direct detection of nucleic acid sequences without biochemical amplification. DNA CT electrochemistry will also be applied as a structural probe in monitoring perturbations in stacking associated with modifications to the DNA bases, as found in DNA lesions, or alterations in the sugar-phosphate backbone. We also propose to apply DNA electrochemistry to probe the kinetics of protein reactions on DNA in real time. We will examine on the millisecond timescale structural perturbations associated with DNA cleavage by the restriction enzyme PvulI and thymine dimer repair by DNA photolyase. Using DNA-modified electrodes, redox cofactors of DNA-binding proteins will also be probed in their DNA-bound form, as will natural product chemotherapeutics which are reductively activated for reaction with DNA. The proposed program therefore intends to exploit DNA-mediated CT in the design of a completely new family of DNA-based sensors forboth fundamental exploration and applied to new technology development. [unreadable] [unreadable]